IgA-seq of human nasalpharyngeal metagenome: 217_presort
Bacteria from human anterior nasal swabs were eluted, stained using FITC-conjugated anti-human IgA, and sorted into IgA-positive and IgA-negative fractions using a Sony MA900 cell sorter. DNA from IgA-positive, IgA-negative and non-sorted fractions was isolated immediately after cell sorting using the Qiagen QIAmp DNA Microbiome kit. DNA was eluted in 50 l of the supplied elution buffer, dried using a SP Genevac miVac centrifugal vacuum concentrator, resuspended in 20 l nuclease-free water and stored at -20C until further use. V1-V3 regions of the 16S rRNA gene were amplified using primers 518F (CAATTACCGCGGCTGCTGG) and 27R (CCGAGTTTGATCMTGGCTCAG), and the pooled amplicon library was sequenced on the Illumina MiSeq platform using MiSeq Reagent Kit v3.